The biological effect of a beef‐derived Latilactobacillus sakei on beef steaks during chilled storage

Abstract The aim of this study was to investigate the biological inhibiting effect of a beef‐derived Latilactobacillus sakei (RS‐25) on the spoilage of beef steaks in overwrapped packaging during the 12 days of storage at 4°C. Beef quality as well as microbial indicators were determined at different intervals during the storage after the inoculation of RS‐25 at the 6 log CFU/g, and the high‐throughput sequencing was applied to investigate the changes of microbial community structure during the storage. The inoculation of RS‐25 on beef had no effect (p > .05) on pH, TBARS, and TVB‐N during storage indicating the weak effect of such strain on the eat quality. Furthermore, the rise of L* and the delayed decline of a* and b* reveal the protection effect of RS‐25 on the meat color. RS‐25 reduced the re‐contaminated Salmonella typhimurium by 1.16 log CFU/g (p < .01), and the growth of Brochothrix thermosphacta was also inhibited but no inhibition was found on the Pseudomonas spp. at the first 6 days of storage. The inhibiting effect of RS‐25 was covered by the rapid growth of other microorganism during the following 6 days of storage. Consistent with the microbial counts results, high‐throughput sequencing analysis confirmed that the inoculated L. sakei RS‐25 was dominant at first 6 days, and then replaced by Pseudomonas spp. The findings obtained from the current study may provide basic information for the further application of bioprotective bacteria in preservation of beef steaks in the overwrapped packaging.


| INTRODUC TI ON
Because of the high nutrient and moisture content, fresh meat is easy to be contaminated by microorganisms, which leads to 21% of total food losses (Jayasena & Jo, 2013;Shao et al., 2021;Slima et al., 2017). Furthermore, bacterial foodborne diseases were major food safety issue, and there is no "single interventionsingle chain point" combination that can completely eliminate the pathogens from the entire beef production chain (Buncic et al., 2014). Therefore, effective antibacterial strategies are needed to inhibit or reduce the survival of microorganisms in fresh beef. Since modern consumers are more inclined to high-quality, healthy, safe, minimally processed, and preservative-free foods (Castellano et al., 2017), lactic acid bacteria (LAB) have been received extensive attention as a natural biological antibacterial agent (Favaro & Todorov, 2017).
Lactic acid bacteria have a long history of safe use and have the qualified presumption of safety (QPS) as determined by the European Food Safety Authority (EFSA) (Koutsoumanis et al., 2020). At present, more than 60 strains of LAB have been recognized by the US Food and Drug Administration (FDA) as generally recognized as safe (GRAS).
LAB are widely used in fermented meat products as starter cultures, and can also be added to meat products as protective cultures without destroying the original quality of the meat products (Barcenilla et al., 2022;Lücke, 1994). For the use in meat products, bioprotective bacteria showed good bacteriostasis effect in sausages (Castilho et al., 2020;Giello et al., 2018;Slima et al., 2017), patties (Castellano et al., 2011;Comi et al., 2015) and ham (Danielski et al., 2020;Hequet et al., 2007), and no negative effects on the shelf life of those products were found. Chen et al. (2020) found that the degree of dominance of Lactobacillus spp. in the beef microflora was considered to be a key factor dictating the shelf-life. Hequet et al. (2007) inoculated cooked ham with 6 log CFU/cm 2 of Latilactobacillus sakei, and the Listeria innocua under the control of L. sakei did not grow significantly throughout the 60-day storage period.
The ability to produce many antibacterial metabolites was the main reason why LAB are used as an antibacterial agent (Leyva Salas et al., 2017). Organic acids such as lactic and acetic acid are the main metabolites of LAB. The low pH environment created by such organic acids is an important factor in inhibiting the growth of undesirable microorganisms (Özcelik et al., 2016). Except organic acids, bacteriocins synthesized by ribosomes of LAB also had a wide range of antimicrobial activities and displayed a target specificity against many strains (Woraprayote et al., 2016). Other metabolites of LAB exhibiting antibacterial effects such as H 2 O 2 , reuterin, and exopolysaccharide were also investigated (Bajpai et al., 2015;Ito et al., 2003;Ortiz-Rivera et al., 2017). Moreover, the competition mechanism exhibited by LAB is also an important antibacterial pathway. It was reported that when LAB was co-cultured with Escherichia coli O157:H7, proteins related to glucose metabolism, energy production, and several other metabolic pathways were over-expressed, and this accelerated their own metabolic rate and improved their ability to compete with other strains for nutritional substrates (Orihuel et al., 2018). This also revealed that LAB may have signal-sensing systems to sense the presence of other microorganisms and respond to stress. Some metabolite of LAB can act as quorum-sensing inhibitors, reducing the release of N-acyl homoserine lactone (AHL) signaling molecules that mediate quorum sensing, thereby inhibiting the formation of pathogenic microbial biofilms (Rana et al., 2020).
Although many factors have the potential capacity to inhibit other microorganism in theory, when it comes to the application, the practical effect depends on many factors such as pH, salt concentration, diffusible water phase, lipid and proteases in meat, and the interactions with other food ingredients or members of the food microbiome (Barcenilla et al., 2022;Blom et al., 1997). The interaction of bacteriocins with lipids in meat, or their inactivation by proteases, will lead to decreased antibacterial ability (Castilho et al., 2020;Favaro & Todorov, 2017). For example, Enterococcus faecalis B1, an antibacterial bacteriocin-producing strain, did not reduce L. monocytogenes by itself in dry-cured ham (Montiel et al., 2019).
Moreover, there are often significant application differences between bioprotective strains. In the study of Iacumin et al. (2021) four bioprotective strains inoculated on cold-smoked sea bass at 5 log CFU/g, and only L. sakei LAK-23 successfully controlled the growth of L. monocytogenes when the storage reached 60 d. Therefore, it is necessary to evaluate the actual application effect of bioprotective candidate strains in meat.
Overwrapped packaging is one of the common meat packaging methods, but the simple packaging leads to a short shelf life. It is particularly important to apply additional food hurdle technology to improve the shelf life of the meat products in this packaging form. The inhibitory effect of LAB on pathogenic bacteria and spoilage bacteria in meat has been preliminarily explored in an aerobic environment and the application potential has been verified. Maragkoudakis et al. (2009) demonstrated that LAB retarded the growth of L. monocytogenes and Salmonella enteritidis in ground chicken within 7 days of storage. Under the same storage condition, Morales et al. (2020) also found the reduced number of Pseudomonas by about 3 log CFU/g by high concentrations of L. sakei on the surface of fresh chicken. Vold et al. (2000) isolated different background flora (about 80% of L. sakei) from ground beef and re-inoculated it into overwrapped ground beef, and found that a high concentration of microbial background effectively inhibited the growth of E. coli O157:H7. At the same time, Danielski et al. (2020) applied Carnobacterium maltaromaticum to overwrapped packaging ready-to-eat meat product, and found it significantly reduced the number of L. monocytogenes by more than 2 log CFU/g compared to the control group after 7 days of storage. Taken together, LAB have an application potential in overwrapped packaged meat products. As far as our knowledge, the application of bioprotective bacteria which isolated from fresh beef and the use of such strain in the following beef cuts preservation is still scarce, and the impact of incubating such strain on the changes of bacterial community structural in beef during the storage of meat needs to be further clarified.
The aim of this study was to clarify the biological protection ability of one beef-derived LAB and its impact on the quality of fresh beef. The quality traits including pH, color, TBARS and TVB-N, and microbial composition including Pseudomonas, B. thermosphacta, Enterobacteriaceae, LAB, and microbial diversity of overwrapped packaged fresh beef when re-contaminated by 6 log CFU/g L. sakei RS-25, were analyzed during 12 days of storage. The inhibitory ability of L. sakei RS-25 against S. typhimurium during storage was also evaluated.

| Antagonistic activity against pathogens
In this experiment, cell-free supernatant (CFS) of L. sakei RS-25 was

| The preparation for the overwrappackaged beef
Four Simmental crossbred cattle of about 24 months were randomly selected in a commercial slaughterhouse (Yangxin, Shandong, China).
After slaughter and chilling in accordance with the GB/T 19477-2018 slaughter operating procedures, the M. longissimus lumborum of the left half carcasses (n = 4) were collected, and then transported to the laboratory under refrigerated conditions for further analysis. Each beef loin was cut into 12 steaks, and then each steak was aseptically cut into five 4 × 4 × 2 cm 3 pieces, and finally all 60 steak pieces were randomly allocated into five storage points in four treatment groups.
In order to test the bioprotective characteristics of L. sakei RS-25 and its effect on beef quality during 12 days of storage, beef samples were re-contaminated with the LAB or sterile water. At the same time, since L. sakei showed the best bacteriostatic effect against S. typhimurium in vitro, it was selected as the indicator bacteria to further study in meat. For the re-contamination, the cells of L. sakei RS-25 were centrifuged at 8000 rpm for 20 min and washed twice with phosphate buffer solution before being collected for use (Slima et al., 2017). Besides control group (G0): No inoculation, sprayed with sterile water, three different treatments were applied as following: LAB treatment group (G1): Beef samples were sprayed with L. sakei RS-25 at 6 log CFU/g; S. typhimurium Group (G2): Beef samples were sprayed with S. typhimurium at 3 log CFU/g; S. typhimurium and L. sakei group (G3): Beef samples were sprayed with S. typhimurium at 3 log CFU/g, dried in a biological safety cabinet for 10 min, and then inoculated with L. sakei RS-25 to a final concentration of 6 log CFU/g. Among them, the quality and spoilage microbial indicators of the G0 and G1 were compared, and the bacterial number of G2 and G3 were counted to evaluate the ability of the RS-25 strain to prevent and control Salmonella spp. in beef.
After the bacterial liquid was absorbed by the meat, the sample was transferred to the overwrapped package tray (TQBC-0775, Oxygen transmission rate at 23°C was 20 cm 3 /m 2 /24 h/0% relative humidity [R.H.]; water vapor transmission rate at 38°C was 15 g/ m 2 /24 h/90% R.H.) (Sealed Air, Shanghai, China), and covered with PE film. The beef samples of each treatment were randomly divided into five time points (0, 3, 6, 9, and 12 days), and quality and microbiological indicators were analyzed at each time point. All samples were stored at 4°C.

| pH determination
The pH value of beef was measured with a pH probe (SenvenGo, Mettler-Toledo, Im Langacher Greifensee, Switzerland) during storage on 0, 3, 6, 9, and 12 days. The pH probe, which was calibrated by buffers with pH 4.00 and 7.00 at room temperature, and the values were recorded on six locations randomly in each sample and the average values were calculated.

| Color measurement
The surface color of steak cuts at 0 d was measured after blooming for 30 min exposed to the air at 0-4°C by using a spectrophotometer (Illuminant A, 4 mm diameter aperture, 10°observer, SP62, X-Rite, Inc., Grand Rapids, USA). Steak cuts at 3, 6, 9, and 12 d were measured immediately after opening the package. Six areas were randomly selected from each cut to determine the lightness (L*), redness (a*), and yellowness (b*). Total color differences (ΔE) were calculated using the following equation: [(L* 1 -L* 0 ) 2 + (a* 1 -a* 0 ) 2 + (b* 1b* 0 ) 2 ] 1/2 , where the subscript 1 represents the LAB treatment group at different time points and the subscript 0 represents the control group.

| Lipid oxidation
Lipid oxidation was assessed by measurement of TBARS using the method of Siu and Draper (1978) with some modification. Samples were taken from the surface of the steaks at each sampling day and stored at −80°C before analysis. Samples (1 g) were transferred to 4 ml of distilled water and ground with a freeze-grinding instrument (Jingxin, Shanghai, China) for 1 min, and then 4 ml of 10% trichloroacetic acid (TCA) was added to the grinding solution. After filtering the grinding solution, 1 ml of the filtrate was taken and 0.25 ml of 0.06 M 2-thiobarbituric acid (TBA) was added, and then incubated at 80°C for 90 min. After that, the absorbance (532 nm) was measured with a microplate reader. Results were expressed as mg malondialdehyde (MDA) equivalent per kg of beef.

| Total volatile basic nitrogen (TVBN)
The concentration of TVBN was determined with reference to the Chinese National Food safety standard method GB 5009.228-2016.
A sub-sample of the steaks was collected at each sampling day and stored at −20°C before analysis. Distilled water (75 ml) was added to ground samples (10 g), with shaking at room temperature and immersion for 30 min. After that, the homogenate was transferred to a distillation tube and 1 g of magnesium oxide was added, which was connected to the automatic Kjeldahl nitrogen determination apparatus (K -355, Buchi, Switzerland), and the volatile basic nitrogen was absorbed by the boric acid solution (20 g/L). Calculated as follows: Where: V1 denotes the volume of hydrochloric acid (ml) consumed by the tested sample. V2 denotes the volume of hydrochloric acid (ml) consumed by the blank sample. c denotes the actual concentration of hydrochloric acid (mol/L). m denotes the weight of the sample (g).

| Microbial analysis
Beef samples (10 g) were collected aseptically from the surface of the steak, and then transferred to stomacher bags (BagPageR®; Interscience, St Nom, France) containing 90 ml of sterile normal saline (1% tryptone and 0.85% sodium chloride), and mixed in a blender (BagMixerR®400; Interscience, St Nom, France). The bacterial solution was serially diluted with 0.1% (w/v) sterile peptone water and the LAB, B. thermosphacta, Enterobacteriaceae, and S. typhimurium in beef were counted at each sampling day during the 12 days of chilled storage.
For the re-inoculation experiment in beef, the cultivation environment of the various microorganisms was as follows: The number of LAB were cultured and counted on MRS medium at 37°C for 48 h. B. thermosphacta was selectively cultured and counted in Steptomycin thallous acetate agar (STAA) (Hope Bio Co., Qingdao, China) with corresponding culture media supplements after incubation at 25°C for 48 h. Enterobacteriaceae was screened and counted on violet red bile glucose agar (VRBGA) (Land Bridge Co., Beijing, China) after incubation at 37°C for 24 h. S. typhimurium was observed and counted on the Salmonella color culture medium (CHROMagar™, France).

| DNA extraction and highthroughput sequencing
Samples (10 g) were taken from the surface of each steak at each sampling day, and the total genomic DNA of the bacteria was extracted (Chen et al., 2020;Yang et al., 2018). Sixty milliliters of liquid from the homogenate in section 2.7 was aspirated, muscle fibers and other tissues were removed through filtration, and then the bacterial cells were collected by centrifugation (10 min, 10,000 rcf). Total genomic DNA was extracted according to magnetic beads method provided by Shannuo™ Bacterial DNA Kit (Tianjing Charme Co., Ltd., China). After DNA concentration and integrity were tested, the 16 S rRNA V3-V4 hypervariable region primers were amplified using 338F (5′−ACTCCTACGGGAGGCAGCA−3′) and 806R (5′− GGACTACHVGGGTWTCTAAT−3′), and the amplified products were purified and quantified.

| Statistical analyses
Two-way analysis of variance (ANOVA) of pH, meat color, TBARS, TVB-N, and microbial counts was carried out using a general linear model in SPSS 26.0 (IBM, USA). The treatment (recontamination or not), storage time and their interaction were fixed factors, and the replicates (loin, n = 4) were the random factor. The data of in vitro antagonistic activity and ΔE were analyzed by one-way ANOVA analysis, in which the indicator bacteria species and storage time were fixed effects, respectively. The effects were compared by LSD method and the differences were considered significant when p < .05.
Community DNA fragments were paired-end sequenced using the Illumina platform, all data were compared with the sliva database. Sequencing raw data were saved in FASTQ format (https:// en.wikip edia.org/wiki/Fastq). Raw sequence data were denoised, spliced, and de-chimerized by DADA2 method (Callahan et al., 2016) with the QIIME2 (2019.4). Based on seed protein sequence of the corresponding functional gene on the RDP website, the insertion and deletion errors in the nucleic acid sequence were corrected by FrameBot (v1.2) (https://github.com/rdpst aff/Framebot). Alpha diversity was used to identify bacterial community richness (Chao1 and Observed species indices), diversity (Shannon and Simpson indices), evenness (Pielou's evenness indices), and coverage (Good's coverage). Hierarchical cluster analysis with the unweighted pairgroup method with arithmetic means (UPGMA) was performed by the uclust function of the R language (version 4.1.0). A heatmap was generated based on the relative abundance of each microbial genus using R script.

| Antagonistic activity against pathogens
In this study, using E. coli O157:H7, L. monocytogenes, S. typhimurium and S. aureus as indicator strains, the in vitro antibacterial effect of L. sakei RS-25 was determined. L. sakei RS-25 has inhibitory effect on all indicator bacteria (inhibition zone > 8 mm), among which the antibacterial effect on S. typhimurium was the best, followed by S. aureus, and finally E. coli O157:H7 and L. monocytogenes (Figure 1).
This was most likely related to the presence of organic acids in CFS.
Decarboxylation systems such as glutamate decarboxylase (GAD) and arginine decarboxylase (ADI) systems exist in E. coli and L. monocytogenes, which consume intracellular hydrogen ions to maintain cellular pH homeostasis (Arcari et al., 2020). However, S. typhimurium lacks GAD system, so there are certain defects in acid resistance (Lin et al., 1995). We should note that the antagonism of LAB against pathogen also involves nutrient competition, and its actual antagonism is also affected by the meat matrix. Therefore, we selected S. typhimurium as the indicator bacteria to further explore the bioprotective effect of L. sakei RS-25 in meat.

| pH
The main effects of storage time, treatment, as well as their interaction had no effect on the pH value of beef samples (p > .05, Table 1). During the chilled storage period for 12 days, the pH value was maintained at the range of 5.51-5.57. This result agrees with the findings of Castellano et al. (2011), who found that after treatment with L. curvatus CRL705 and Lactococcus lactis CRL1109, the pH value of beef steaks did not change significantly over 9 days of storage. Another study conducted by Danielski et al. (2020) also reported that over 7 days after the ham surface was inoculated with C. maltaromaticum, no significant changes in pH value were found.
However, a study reported that after a storage period of 38 days under vacuum conditions, L. sakei and L. curvatus significantly reduced the pH of fresh beef to 5.27 and 5.34, respectively .
This may be related to the accumulation of acid production due to longer storage time in the vacuum condition compared with the current study, and the fact that LAB can produce organic acids to reduce the pH. Jones et al. (2010) also found that the metabolites of L. sakei reduced the surface pH value of the lamb after 6 weeks.
LAB are commonly used as a starter and added to fermented meat products, usually reducing the pH of the fermented meat to protect product safety. However, LAB acting as protective cultures (rather than starter cultures) should not alter the flavor and appearance of the product, especially the large drop in pH (Lücke, 1994).

| Meat color
The effect of the re-contamination of L. sakei RS-25 on the color of beef during chilled storage is shown in Table

| Lipid oxidation (TBARS) and TVB-N
The detection of TBA is widely used in the evaluation of lipid oxidation in meat products, and TBARS value is one of the important indicators reflecting the quality of beef (Sun et al., 2001).
Despite of incubating L. sakei RS-25 or not, the storage time escalated the degree of lipid oxidation of beef samples (p < .05; Table 1). However, one noticeable point in this study is that the recontamination of L. sakei (6 log CFU/g) on the beef surface did not increase the TBARS values than the control in each sampling day.  This indicated that the inoculated LAB did not aggravate the lipid oxidation during beef storage, which is consistent with the reports of Slima et al. (2017), who found that large number LAB (7 or 8 log CFU/g) as protective cultures did not aggravate lipid oxidation in fresh beef sausages.
A similar trend that the storage time increased the TVB-N values despite of incubating L. sakei RS-25 or not was also investigated (p < .05; Table 1). On the 9th day of storage, the control group and the LAB treatment group reached 13.3 mg/100 g and 14.0 mg/100 g, respectively, which were close to the spoilage limit standard value  (Table 1). These results were in accordance with the findings previously reported by Zhang et al. (2018) showing that after the steak surface was treated with 7 log CFU/g of L. sakei or L. curvatus, no increase in TVB-N values was observed during storage for 4 weeks. In general, LAB have a minimal impact on meat quality, suggesting that even its large presence does not lead an acceleration of the lipid oxidation or meat spoilage. Table 2 shows the effect of inoculating L. sakei RS-25 on the reduction of the bacteria in beef steak. The inherent number of LAB in the control group was 4.13 log CFU/g, which was higher than the two abattoirs in China investigated by Chen et al. (2020). In the G1 treatment group, after being inoculated with L. sakei RS-25, the number of LAB reached 6 log CFU/g. Although L. sakei RS-25 colonized in beef steaks successfully, no significant growth occurred during the first 6 days. At the end of storage (12 days), the number of LAB in the G1 G0 groups increased to 7.28 log CFU/g and 5.09 log CFU/g, respectively. Compared with the control group, L. sakei RS-25 significantly reduced a certain number of spoilage bacteria within 6 days (Table 2), for instance, the population of B. thermosphacta was reduced 0.38 log CFU/g by 6 d of storage and S. typhimurium was also reduced 0.59 log CFU/g and 1.16 log CFU/g after 3 and 6 d of storage, respectively, for re-contaminated steaks (Table 2, p < .05). Furthermore, the number of S. typhimurium did not increase (p > .05) within the first 6 days of storage compared to the control group (Table 2). There was no significant difference in the colony count of Pseudomonas between the two groups for the first 6 days of storage which indicating LAB had no inhibitory effect on Pseudomonas during such a storage period.

| Microbial counts
The inhibitory effect of L. sakei RS-25 on B. thermosphacta and S. typhimurium may be the result of antagonistic effect among L. sakei and other autochthonous bacteria in raw meat such as the competition for nutrients, oxygen or hydrogen sources (Nychas et al., 2008). Effective utilization of nutrients such as glucose and glutamine by LAB can control the growth of spoilage bacteria (Honoré et al., 2016). Under the growth conditions of this study, the nutrient competition ability produced by 6 log CFU/g of L. sakei TA B L E 2 Effect of the re-contamination of lactic acid bacteria on the microbial loads in beef during chilled storage up to 12 days consist of the assumption of the Jameson effect that the dominating microbiota quantitatively inhibits growth of the pathogen in the same way that they inhibit their own growth, and the pathogen therefore stops growing at the time when the dominating microbiota reach their maximum population density. Other studies also find the same phenomenon, whether the original beef microflora consisted mainly of LAB of which approximately 80% were L. sakei (Vold et al., 2000) or a single strain with bacteriostatic properties (Danielski et al., 2020) was used as a high-concentration microbial background, its effective prevention and control of pathogenic microorganisms in overwrapped packaging was confirmed. Other reports also confirmed that high concentrations of microflora in raw meat inhibited the growth of Salmonella spp. (Moller et al., 2013;Zaher & Fujikawa, 2011).
Latilactobacillus sakei showed bioprotective potential within 6 days, however not as effective as applications in vacuum packaging. In fact, the other microorganisms grew significantly after 6 d of storage in this study (Table 2), at which point the inhibitory ability of LAB disappeared. LAB inoculated on vacuum-packed steaks by Zhang et al. (2018) extended the shelf life of steaks by at least 10 days. Vold et al. (2000) compared the application effects of high-concentration background microorganisms between vacuum and overwrapped packaging, and also found that L. sakei had bacteriostatic effects in both packaging situations, and the inhibition was more pronounced in vacuum packaging. As mentioned before, L. sakei RS-25 did not significantly reduce the pH value in the current condition and this may weaken the inhibiting effect of LAB in a large extent. The inhibition of other bacterial species may be an advantage in the number of LAB. Morales et al. (2020) observed the inhibition of Pseudomonas by 8 log CFU/g LAB inoculum on chicken surfaces, which may be due to the stronger nutrient competition potential of higher initial LAB numbers. Other researchers argued that the ability of LAB to produce available metabolites is an important factor in their antibacterial properties (Ioannis et al., 2014). Bolívar et al. (2021) observed the inhibitory effect on L. monocytogenes when L. sakei reached the maximum population density, and they believed that bacteriocin may play a special antagonistic effect at this time. However, it may be difficult for LAB to metabolize sufficient levels of bacteriostatic substances in meat compared to in pure culture medium, and the presence of these bacteriostatic substances such as bacteriocins in meat has not been quantified in relevant studies (Bolívar et al., 2021;Costa et al., 2019). Morales et al. (2020) found that the antibacterial effect of L. sakei on Pseudomonas spp. in actual application on fresh chicken was far less than that in pure culture medium. Therefore, whether the inhibitory effect is due to metabolites or bacterial population remains to be further explored.
It was also found in the current study that the storage period of 6 d seems to be a critical time point (Table 2). After 6 days, the number of most spoilage bacteria had exceeded 6 log CFU/g, which was also the initial inoculum of L. sakei. It is worth noting that B. thermosphacta, Pseudomonas spp., and S. typhimurium had rapid growth after the 6th day of storage in the presence of lactic acid bacteria inoculation (G1), and the growth rate exceeded that of the control group (G0). This may be related to the synergistic effect of Pseudomonas spp. with other microorganisms. Pseudomonas have a strong ability to hydrolyze carbohydrate and lipids, which would made more readily available sources of carbohydrates. On the other hand, the hydrolysis of proteins by Pseudomonas can release peptides and amino acids (Marshalld & Schmidtr, 1991). The more readily available nutrients create favorable conditions for the growth of other spoilage and pathogenic bacteria, even LAB themselves.
Due to the lower spoilage ability of L. sakei RS-25 in this study, and the 6 log CFU/g microbial background provided in the meat, the above data suggest that the microbial background has a certain protective effect in the first 6 days. However, L. sakei RS-25 lost its dominant position in the later period, especially after the rapid growth of and 7.74% on 0, 3, and 6 d, respectively). After 6 days of storage, the beef microflora was completely dominated by Pseudomonas spp., and these results were consistent with the microbial count results, which proved that the beef had tended to spoil at this time.
Different processing environments affect the initial microflora of beef. Chen et al. (2020) investigated beef bacterial composition from two abattoirs and found that the environment of abattoir B was closer to the beef processing environment in this study, and Pseudomonas spp. (31.4%), Acinetobacter spp. (17.2%), and Psychrobacter spp.
(10.1%) were the predominant bacteria. In this study, Pseudomonas was the dominant bacteria, although the relative abundance of Pseudomonas spp. in the treatment group (G1) was only 1.39%, but this did not affect its later development as a dominant spoilage strain (Figure 3b). Recent work has shown that Pseudomonas is more likely to become the dominant spoilage bacteria in oxygen permeable environment (Mansur et al., 2019;Pennacchia et al., 2011;Wang et al., 2018). Spoilage Pseudomonas spp., grown on chilled meats, has a range of properties that aid their survival under low temperatures, giving it a competitive advantage. Pseudomonas have strong spoilage ability due to a capacity to produce proteolytic and lipolytic enzymes (Remenant et al., 2015;Stanborough et al., 2018).
When the glucose and lactic acid in the meat are depleted, it secretes extracellular proteases that break down the connective tissue between muscle fibers, allowing bacteria to penetrate the muscle tissue (Wickramasinghe et al., 2019). Gupta and Nagamohini (1992) study showed that at 37°C, Pseudomonas spp. was able to penetrate 3 cm in meat after 24 h, and its penetration in muscle was far greater than that of Lactobacillus spp., reflecting the differences in the nature and extent of protease production between Pseudomonas and Lactobacillus. And, even at low cell numbers, psychrotrophic spoilage pseudomonads is capable of secreting cold-active proteases and lipases, and at temperatures below the optimum growth temperature, it produces more lipases (Wickramasinghe et al., 2019).
Both the siderophores' production capacity and efficient utilization rate of glucose make Pseudomonas superior in the competition for nutrients (Gram et al., 2002;Tsigarida et al., 2003). In addition, its low oxygen affinity and effective competition for iron make it easier for Pseudomonas to dominate meat foods stored aerobically during chilled storage (Ellis et al., 2000;Mohareb et al., 2015). On the contrary, there is only a small amount of glucose and glucose-6-phosphate in lean meat (Lücke, 1994), so that LAB do not have F I G U R E 2 Richness diversity estimator of bacterial communities in overwrapped packaged beef during chilled storage up to 12 days. C -control, without re-contamination (G0); L -beef samples re-contaminated by L. sakei RS-25 with the initial number of LAB reached 6.0 log CFU/g (G1) enough fermentable sugars, which reduces the lactic acid and other metabolites of LAB and affects its antibacterial effect.
The heatmap was used to describe the relative abundance changes of the top 20 genera in 12 days, and the cluster tree was drawn above the graph by unweighted pair-group method with arithmetic (UPGMA) means according to the abundance similarity and spoiled (C_9d, C_12d, L_12d). It is worth noting that the microflora structure of the LAB treatment group on the 9th day (L_9d) was more similar to the fresh one. This revealed that the presence of L. sakei RS-25 delayed the changes of microflora structure.
For principal component analysis (PCA), the maximum variations of microbiota in beef samples were 81.6% (PC1) and 18.4% (PC2), representing a strong regional separation (Figure 4b). We clearly observed that control group (G0) on 6 d was clustered with 9 and 12 d of the two groups (G0 and G1), which also illustrated that its microbial composition was more similar. On the contrary, the treatment group on 6 d deviated from the completely spoiled microflora structure. The presence of LAB reduced the diversity and richness of beef microflora and optimized the microflora structure to a certain F I G U R E 3 Relative abundance of the bacteria at the phylum level (a) and genus level (b) in overwrapped packaged beef during chilled storage up to 12 days. C -No inoculation treatment (G0); L -beef samples were treated with L. sakei RS-25 at 6 log CFU/g (G1) extent. Chen et al. (2020) also speculated that the dominance of Lactobacillus spp. was suggested an important factor affecting shelf life. However, as analyzed above, L. sakei did not effectively control the growth of Pseudomonas spp. maybe due to the metabolic environmental factors (Morales et al., 2020). Although L. sakei did not extend the shelf life of beef in overwrapped packaging, it delayed the succession process of the beef microflora structure to bacteria with high spoilage potential. At last, it should be noted that some L. sakei strains can break down amino acids in meat to produce unpleasant flavors. Leisner et al. (1996) reveal that one L. sakei 1218 strain developed a distinct sulfide odor after 3 weeks of storage in vacuum-packed beef and methods should be employed to inhibit the growth of such specific strain. Therefore, to be successful in bioconservation, it is necessary to further explore the interspecific diversity of LAB during the storage, especially for the ones who have the ability to decompose fat and protein in meat.

F I G U R E 4
Community heatmap analysis (a) and principal component analysis (PCA; b) at the genus-level of different microbiota in overwrapped packaged beef during chilled storage up to 12 days. C -No inoculation treatment (G0); L -beef samples were treated with L. sakei RS-25 at 6 log CFU/g (G1).

| CON CLUS IONS
This study concluded that the addition of L. sakei RS-25 as a protective culture to fresh beef inhibited the growth of S. typhimurium and B. thermosphacta within 6 days of storage. In addition, RS-25 had no negative effects on beef pH, TBARS, and TVB-N, and also delayed the deterioration of meat color. Furthermore, the microbial composition analysis indicated that RS-25 had an obvious optimization effect on the microbial flora structure of fresh beef, which could delay the evolution trend of microbial flora to spoilage flora. Nevertheless, further studies should consider the practical application of meat substrate, packaging environment, and other factors on the impact of bioprotective bacteria, and to select suitable initial bacteria quantity, or combine with other antibacterial technologies to achieve a better antibacterial effect.

ACK N OWLED G M ENTS
This work was financially supported by a special intergovernmental project for international cooperation in Science, Technology, and Innovation (2019YFE0103800), China agriculture research system of MOF and MARA (CARS-37), and Special fund for innovation team of modern agricultural industrial technology system in Shandong Province (SDAIT-09-09).

CO N FLI C T O F I NTE R E S T
No conflict of interest exits in the submission of this manuscript, and this manuscript has been approved by all authors for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request.